CN111359263A - Capillary coated column for chiral compound separation, preparation method and application thereof - Google Patents
Capillary coated column for chiral compound separation, preparation method and application thereof Download PDFInfo
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- CN111359263A CN111359263A CN202010099207.4A CN202010099207A CN111359263A CN 111359263 A CN111359263 A CN 111359263A CN 202010099207 A CN202010099207 A CN 202010099207A CN 111359263 A CN111359263 A CN 111359263A
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Abstract
The invention discloses a capillary coated column for separating chiral compounds, wherein the inner wall of a capillary is modified with a vancomycin covalent bonding coating, the vancomycin covalent bonding coating is formed by light-sensitive diazoresin and vancomycin through ultraviolet irradiation to perform photocuring crosslinking reaction, the thickness of the vancomycin covalent bonding coating is 1-10nm, and the coating is formed by light-sensitive diazoresin and vancomycin through ultraviolet irradiation to perform photocuring crosslinking reaction. The capillary tube coating prepared by the invention has the advantages of simple and rapid preparation process, good hydrophilicity and excellent performance.
Description
Technical Field
The invention relates to the field of compound separation, in particular to a capillary coated column for chiral compound separation, and also relates to a preparation method of the capillary coated column for chiral compound separation and application of the capillary coated column in the chiral compound separation process.
Background
The separation of chiral compounds is becoming increasingly important in many industries, particularly the pharmaceutical industry, and this interest is attributed to the different pharmacokinetic profiles and pharmacological activities of each enantiomer in racemic drugs. Enantiomeric separations have used many different types of instruments and methods, including TLC, HPLC, GC, and CE, among others. The TLC separation of chiral compounds is simple and convenient to operate, simple in equipment, high in analysis speed and visual in result. But the separation dosage is larger, and the loss amount is large in the separation process. The separation modes of HPLC include a chiral stationary phase method, a chiral mobile phase additive method and a chiral reagent derivation method. The chiral stationary phase method needs a special stationary phase, the price is high, a small amount of samples still need pre-column derivatization, and because a chromatographic column is short and the column efficiency can only reach thousands, certain drug enantiomers cannot be separated; the compounds which can be resolved by the chiral mobile phase additive method are limited, and the properties of individual chiral additives are not stable enough and cannot be separated; the chiral reagent derivatization method requires a high purity derivatization reagent and the rate of derivatization between the two enantiomers cannot be guaranteed. The GC method has high separation efficiency, is more suitable for the resolution of chiral drugs with low boiling points and easy gasification, but has few drugs with the conditions, and the drugs are easy to isomerize by high-temperature analysis.
Chiral compounds, although having very similar chemical structures, can interact differently with peptides, proteins, enzymes. For example, for several chiral drugs, the pharmacological activity of both enantiomers is higher than the others, but in some cases one of the two compounds may even be harmful to health. Therefore, there is an urgent need to use more advanced and reliable assay methods for determining enantiomeric compounds present in different substrates (e.g., pharmaceutical preparations, biological samples, forensic, environmental, agricultural and food samples).
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is that the existing method for analyzing the chiral compound by using the capillary coated column has the problems of large sample consumption and loss, high cost, long time consumption, low separation efficiency and long analysis time. The capillary tube with the vancomycin diazo polymer coating can realize the advantages of high separation speed and more thorough separation between chiral compounds when the capillary tube is used for separating the chiral compounds.
In order to solve the technical problems, the invention adopts the following technical scheme:
a capillary coated column for chiral compound separation comprising the steps of:
the inner wall of the capillary is modified with a covalent bonding coating of vancomycin, the covalent bonding coating of vancomycin is formed by light-cured crosslinking reaction of photosensitive diazo resin and vancomycin through ultraviolet irradiation, and the thickness of the covalent bonding coating of vancomycin is 1-10 nm.
The preparation method of the capillary coated column for chiral compound separation comprises the following steps:
s1, concentration (2-6) mg-1The photosensitive diazo resin solution of (2-6) mg.mL of water-1The vancomycin solution and water are washed into the activated capillary tube in sequence; the dosage ratio of the photosensitive diazoresin to the vancomycin is 1: (1-1.8);
s2, exposing the capillary tube coating column prepared in the step S1 under an ultraviolet lamp with the wavelength of 248nm-365nm for 15min-30min, wherein the exposure dose is 5-20000mJ/cm2And obtaining the capillary column with the diazo resin/vancomycin coating.
The step S1 is performed at a temperature of 10 to 50 ℃.
The weight average molecular weight Mw of the photosensitive diazo resin is 750-5000, and the molecular weight Mw of the vancomycin is 1000-3000.
The step S1 is repeated 1-8 times, preferably 1-2 times.
A capillary tube for chiral compound separation prepared by the method.
The application of the capillary tube for chiral compound separation in chiral compound separation.
Compared with the prior art, the invention has the following beneficial effects:
(1) the chirality separated capillary coated column prepared by the method can be used for the occasions of electrophoretic separation detection and purification of chiral samples and the like. Capillary Electrophoresis (CE) is a separation technique commonly used in analysis due to rapid separation, and compared with Liquid Chromatography (LC), has short analysis time, high efficiency, small reagent consumption and minimal sample consumption; the capillary coating can allow fine adjustment of electroosmotic flow (EOF), diazo resin is selected to replace a coupling agent silanization reagent with high toxicity and high price, and the diazo resin and vancomycin are subjected to light curing crosslinking reaction through ultraviolet irradiation to form a covalent bonding coating, so that the problems of complex process flow, harsh operating conditions, high toxicity, high production cost, easiness in causing nonuniformity and irregularity of the coating, even capillary blockage and the like in the preparation process of the capillary electrophoresis coating column are solved; the selectivity and separation effect of CE are also improved.
(2) By adopting the method, the photosensitive diazoresin is used for replacing a silanization reagent with high price and high toxicity for bonding the vancomycin, the preparation process is simple and convenient, the cost is low, the production process is green and environment-friendly, and the separation reproducibility is good.
(3) Advantages of the capillary column of the present invention in the separation of chiral compounds
The capillary tube with the vancomycin diazo polymer coating can realize the advantages of high separation speed and more thorough separation between chiral compounds when the capillary tube is used for separating the chiral compounds.
Drawings
FIG. 1 is a comparative analysis chart of the separation performance of the chiral substance chlorpheniramine maleate in example 1
FIG. 2 is a comparative analysis chart of the electrophoretic separation performance of the benzoin of the chiral substances in example 1.
FIG. 3 is a comparative analysis chart of the electrophoretic separation performance of the chiral substance promethazine in example 1.
FIG. 4 is a schematic diagram of a synthesis process for a chirally separated capillary coated column of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.
The invention provides a capillary coated column for chiral compound separation, which comprises the following steps:
the inner wall of the capillary is modified with a covalent bonding coating of vancomycin, the covalent bonding coating of vancomycin is formed by light-cured crosslinking reaction of photosensitive diazo resin and vancomycin through ultraviolet irradiation, and the thickness of the covalent bonding coating of vancomycin is 1-10 nm.
The preparation method of the capillary coated column for chiral compound separation comprises the following steps:
s1, concentration (2-6) mg-1The photosensitive diazo resin solution of (2-6) mg.mL of water-1The vancomycin solution and water are washed into the activated capillary tube in sequence; the dosage ratio of the photosensitive diazoresin to the vancomycin is 1: (1-1.8);
s2, exposing the capillary tube coating column prepared in the step S1 under an ultraviolet lamp with the wavelength of 248nm-365nm for 15min-30min, wherein the exposure dose is 5-20000mJ/cm2And obtaining the capillary column with the diazo resin/vancomycin coating.
The step S1 is performed at a temperature of 10 to 50 ℃.
The weight average molecular weight Mw of the photosensitive diazo resin is 750-5000, and the molecular weight Mw of the vancomycin is 1000-3000.
The step S1 is repeated 1-8 times, preferably 1-2 times.
A capillary tube for chiral compound separation prepared by the method.
The application of the capillary tube for chiral compound separation in chiral compound separation.
The separation performance of the chiral substance is measured by a CE-7100 type capillary electrophoresis apparatus, the concentration of an analyte (chlorpheniramine, benzoin or promethazine) is 0.5mg/ml, the ultraviolet detection wavelength is 214nm, the effective lengths of a covalent bonding coating column and a quartz capillary bare column are 41cm, the inner diameter is 50 mu m, the column temperature is 25 ℃, the separation voltage is +18kV, and the separation medium is a TEAA/ACN solution with the concentration of 40 mM.
The invention has the following embodiments: example 1 will be described in further detail with reference to examples
Reagent: 0.1mol/L NaOH solution (strong alkaline solution), 0.1mol/L HCl solution, 2 mg/ml -12 mg/ml-1 vancomycin solution and 40mM TEAA/ACN solution;
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol sequentially for 25min, 12min, 25min, 10min and 10min, respectively, and blow-drying the capillary tube with inert gas to obtain an activated capillary tube;
s1, at 10 deg.C, using the concentration of 2 mg/ml-1The photosensitive diazoresin solution washes the capillary column for 10min, and the distilled water washes the capillary column for 2min, so that the capillary is prevented from being blocked due to the overlarge concentration of the buffer solution; and then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 2 mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 1 time.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 325nm for 15min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 1-layer composite structure.
The covalently bonded coating has excellent chiral separation performance due to shielding by photocuring crosslinking reaction between the silicon hydroxyl groups on the inner wall of the capillary and the coating. FIG. 4 is a schematic diagram showing the synthesis of a chiral separation capillary coating column according to the present invention, wherein the upper circle is a cross-sectional view of the capillary, the lower circle is a cross-sectional view of the capillary, the black squares in the figure represent the silicon hydroxyl groups on the inner wall of the capillary, the triangles in the figure represent the photosensitive diazo resin, and the circles represent vancomycin. FIG. 4 shows the reaction from left to right, in which the photosensitive diazo resin is bonded to the silicon hydroxyl groups on the inner wall of the capillary tube, and acts on the wall of the capillary tube in an electrostatic adsorption manner; the photosensitive diazoresin and the vancomycin react under the condition of no illumination, namely diazoresin ionizes in aqueous solution to form diazo groups which are combined with hydroxyl carried by the vancomycin through the action of ionic bonds; the photosensitive diazo resin and the vancomycin have photocuring crosslinking reaction, namely under the irradiation of ultraviolet light, diazo groups are decomposed, so that ionic bonds between the diazo resin and the vancomycin are converted into more stable covalent bonds.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, repeat the experiment 5 times under the above conditions, and calculate the relative standard deviation of peak 1 and peak 2.
FIG. 1 is a graph showing comparative analysis of the separation performance of a capillary tube of the present invention on a chiral substance chlorpheniramine, FIG. 2 is a graph showing comparative analysis of the separation performance of a capillary tube of the present invention on a chiral substance benzoin, and FIG. 3 is a graph showing comparative analysis of the separation performance of a capillary tube of the present invention on a chiral substance promethazine. As can be seen from FIG. 1, the separation can be achieved within a short 16min period, and the separation between the peaks of the electrophoretic separation curves of the two enantiomers is large, so that the capillary with the vancomycin diazo polymer coating provided by the invention can more thoroughly separate the chiral substances;
the separating mechanism is illustrated by promethazine as an example: the promethazine and the hydroxyl in the vancomycin act simultaneously, an amino group contained in the promethazine and the hydroxyl in a vancomycin fixed phase react to form a hydrogen bond, and the bonding force of the hydrogen bond formed by the levorotatory promethazine and the dextrorotatory promethazine and the hydroxyl of the vancomycin is different, so that the retention time of the levorotatory promethazine and the dextrorotatory promethazine is different from that of the hydroxyl of the vancomycin, and then two enantiomers are separated by chromatographic elution.
As can be seen from FIG. 2, the electrophoretic separation curves of the capillary coated column and the bare capillary are different, and by using the capillary coated column for chiral separation of the invention, the two enantiomers of benzoin are separated within 12 min; as can be seen from FIG. 3, the separation can be achieved within 15min, the interval between peaks is large, and the peak width is also large, so that the capillary tube with the vancomycin diazo polymer coating provided by the invention can achieve a good separation effect on the chiral substances;
example 2
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 3 mg/ml-1The photosensitive diazo resin solution, 6 mg/ml-1 vancomycin aqueous solution and 40mM TEAA/ACN solution;
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 35min, 8min, 30min, 8min and 12min, respectively, and blow-drying the capillary tube with inert gas to obtain an activated capillary tube;
s1, at 10 deg.C, using the concentration of 3 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 6 mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 2 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 300nm for 20min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 4-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.28% and 0.521%, respectively.
Example 3
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 4 mg/ml-1The photosensitive diazo resin solution, a vancomycin aqueous solution of 3 mg/ml < -1 >, a TEAA/ACN solution with the concentration of 40 mM;
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 30min, 10min, 35min, 12min and 8min, respectively, and blow-drying the capillary tube with inert gas to obtain an activated capillary tube;
s1, under the condition of 10 ℃, the concentration is 4 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 3 mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 3 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 280nm for 15min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 6-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the example of a chlorpheniramine sample, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.352% and 0.697%, respectively.
Example 4
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 5mg/ml -14 mg/ml-1 vancomycin solution and 40mM TEAA/ACN solution;
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min, respectively, and blow-drying the capillary tube with inert gas to obtain activated capillary tube
S1, at 10 deg.C, using 5mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a 4 mg/ml & lt-1 & gt vancomycin aqueous solution, and growing along the inner wall of the quartz capillary under the action of the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 4 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 260nm for 18min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 8-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.437% and 0.78%, respectively.
Example 5
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 6 mg/ml -12 mg/ml-1 of vancomycin solution at a concentration of 40mMThe TEAA/ACN solution of (1);
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min, respectively, and blow-drying the capillary tube with inert gas to obtain activated capillary tube
S1, under the condition of 10 ℃, the concentration is 6 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 2 mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 5 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 250nm for 30min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 10-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.51% and 0.83%, respectively.
Example 6
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 2 mg/ml-1The photosensitive diazo resin solution, a vancomycin aqueous solution of 3 mg/ml < -1 >, a TEAA/ACN solution with the concentration of 40 mM;
s0, capillary tube internal activation pretreatment
Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min, respectively, and blow-drying the capillary tube with inert gas to obtain activated capillary tube
S1, at 10 deg.C, using the concentration of 2 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 3 mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 2 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 320nm for 20min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 4-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.27% and 0.45%, respectively.
Example 7
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 2 mg/ml -14 mg/ml-1 vancomycin solution and 40mM TEAA/ACN solution;
at 10 deg.C, the concentration is 2 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a 4 mg/ml & lt-1 & gt vancomycin aqueous solution, and growing along the inner wall of the quartz capillary under the action of the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 3 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 300nm for 25min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 6-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.35% and 0.497%, respectively.
Example 8
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 3 mg/ml-1The photosensitive diazo resin solution, 5 mg/ml-1 vancomycin aqueous solution and 40mM TEAA/ACN solution;
at 10 deg.C, the concentration is 3 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 5mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 4 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 340nm for 18min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 8-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.42% and 0.573%, respectively.
Example 9
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 2 mg/ml-1The photosensitive diazo resin solution, 5 mg/ml-1 vancomycin aqueous solution and 40mM TEAA/ACN solution;
at 10 deg.C, the concentration is 2 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. And then washing the capillary column for 10min by using a vancomycin aqueous solution with the concentration of 5mg/ml & lt-1 & gt, and growing along the inner wall of the quartz capillary under the driving force of the vancomycin diazo polymer solution injector to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 5 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 300nm for 21min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 10-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.49% and 0.632%, respectively.
Example 10
Reagent, 0.1mol/L NaOH solution, 0.1mol/L HCl solution, 2 mg/ml-1The photosensitive diazo resin solution, 6 mg/ml-1 vancomycin aqueous solution and 40mM TEAA/ACN solution;
at 10 deg.C, the concentration is 2 mg/ml-1The capillary column is washed by the photosensitive diazo resin solution for 10min, and the capillary column is washed by distilled water for 2 min. The capillary column was then rinsed for 10min with an aqueous vancomycin solution having a concentration of 6mg ml-1 and the vancomycin diazopolymer solution syringeAnd growing along the inner wall of the quartz capillary tube under the action of the driving force to finish the self-assembly process. Then, the capillary column is washed by distilled water for 1 minute, so that 1 electrostatic self-assembly cycle is completed; this step was repeated 6 times.
And S2, exposing the capillary tube prepared in the step S1 under an ultraviolet lamp with the wavelength of 280nm for 15min to construct a covalently bonded capillary column with a diazo resin-vancomycin composite 12-layer composite structure.
S3, taking the concentration of the analyte as 0.5mg/ml, detecting in a TEAA/ACN solution with the ultraviolet detection wavelength of 214nm, the quartz capillary bare column inner diameter of 50 μm, the column temperature of 25 ℃, the separation voltage of +18kV and the separation medium concentration of 40 mM.
S4, taking the chlorpheniramine sample as an example, the experiment was repeated 5 times under the above conditions, and the relative standard deviations of peak 1 and peak 2 were 0.57% and 0.75%, respectively.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (9)
1. A capillary coated column for chiral compound separation is characterized in that,
the inner wall of the capillary is modified with a covalent bonding coating of vancomycin, the covalent bonding coating of vancomycin is formed by light-cured crosslinking reaction of photosensitive diazo resin and vancomycin through ultraviolet irradiation, and the thickness of the covalent bonding coating of vancomycin is 1-10 nm.
2. A method of preparing a capillary coated column for chiral compound separation according to claim 1, comprising the steps of:
s1, concentration (2-6) mg-1Is light-sensitiveAzosin solution, water, concentration (2-6) mg.mL-1The vancomycin solution and water are washed into the activated capillary tube in sequence; the dosage ratio of the photosensitive diazoresin to the vancomycin is 1: (1-1.8);
s2, exposing the capillary tube coating column prepared in the step S1 under an ultraviolet lamp with the wavelength of 248nm-365nm for 15min-30min, wherein the exposure dose is 5-20000mJ/cm2And obtaining the capillary column with the diazo resin/vancomycin coating.
3. The method as claimed in claim 2, wherein the step S1 is performed at a temperature of 10 to 50 ℃.
4. The method as claimed in claim 3, wherein the weight average molecular weight Mw of the photosensitive diazo resin is 750-5000, and the molecular weight Mw of the vancomycin is 1000-3000.
5. The method of claim 4, wherein the step S1 is repeated 1-8 times.
6. The method of claim 5, wherein the step S1 is repeated 1-2 times.
7. A capillary tube for separation of chiral compounds prepared by the method of any one of claims 1 to 6.
8. Use of the capillary for chiral compound separation according to claim 7 for separation of chiral compounds.
9. The use according to claim 8, wherein the chiral compound is chlorpheniramine maleate, benzoin, promethazine.
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